A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.
Day 1: F. tularensis plate inoculation
Day 2: F. tularensis liquid media inoculation
Day 3: Spheroplasting, osmotic lysis, and sucrose density gradient centrifugation
Day 4: Collection of sucrose gradient fractions
Representative Results
When performed correctly, this procedure results in the complete separation of IM and OM vesicles from both Type A (e.g. SchuS4) and Type B (e.g. LVS) strains of F. tularensis. As shown in representative immunoblots in Figure 1, F. tularensis OMPs localize between densities of 1.17 and 1.20 g/ml. By comparison, IMPs localize between densities of 1.13 and 1.14 g/ml.
Figure 1. Immunoblotting of F. tularensis sucrose density gradients. Sequential fractions were collected from gradients and densities (g/ml) were calculated based upon refractive indices. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted to detect OMP and IMP fractionation. mw, prestained molecular weight standards with sizes (in kDa) noted on the left side of each blot. LVS, whole cell lysates of F. tularensis LVS. The corresponding sucrose gradient fraction densities are noted above their respective lanes. OMPs and IMPs, as noted in the left margin, were detected with polyclonal, monospecific antisera. Similar results were obtained for F. tularensis SchuS4.
This protocol describes a variation of the spheroplasting, osmotic lysis, and sucrose density gradient centrifugation “gold standard” for the physical separation and enrichment of Gram-negative bacterial OMs from other cellular components 2, 3, 4. Whereas we previously described the application of this method for isolating OMs from both Type A and Type B strains of F. tularensis 5, this presentation offers a more detailed and optimized procedure for OM isolation. Indeed, this is an important advance for identifying potentially surface-exposed and presumptive virulence factors from this deadly pathogen. The significance of this procedure was demonstrated in studies by our lab showing that immunization of mice with purified OMPs afforded substantial protection against virulent Type A F. tularensis pulmonary challenge 6. As noted above, growth phase of the bacterial cells, careful monitoring during osmotic lysis, and gentle resuspension of membrane pellets are critical steps that appear to correlate with success/failure of this membrane separation procedure. While this optimized method is rigorous and subject to some experimental variability, the resulting OMs appear to be of remarkably enhanced purity as compared to those garnered from the use of detergents, lithium chloride, or sodium carbonate 7, 8, 9, 10, 11, 12.
The authors have nothing to disclose.
The project described was supported by Grant Numbers P01 AI055637 and U54 AI057156 from NIAID/NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the RCE Programs Office, NIAID, or NIH.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Donor calf serum | Mediatech | 35-022-CV | ||
IsoVitaleX | Becton Dickinson | 211876 | ||
Cellgro molecular grade dH2O | Mediatech | 46-000-CM | ||
38.5 ml polycarbonate bottle | FiberLite | 010-0514 | ||
F50L-8×39 ultracentrifuge rotor | FiberLite | 096-087051 | ||
Complete mini EDTA-free protease inhibitor cocktail | Roche Diagnostics | 04693132001 | ||
Benzonase | Novagen | 70664-3 | ||
DC protein assay | BioRad | 500-0116 | ||
Ultra-Clear 14x95mm centrifuge tubes | Beckman | 344060 | ||
SW-40Ti swinging bucket rotor | Beckman Coulter | 331301 | ||
Abbe benchtop refractometer | Fisher Scientific | 13-964-10 |